Project Summary/Abstract Most of the sporadic colon cancer in this country arises from genetic mutations that activate the canonical WNT/beta-catenin signaling pathway. Yet in the clinic, colon cancer patients are treated the same way they have been for decades: a chemotherapy regimen (e.g. FOLFOX for stages III, IV) and an add-on anti- angiogenic agent such as Bevacizumab or Avastin. Add-on treatments extend life for ~5 months, but they almost always fail because tumors develop resistance. Only one Wnt inhibitor has made it to a Phase II clinical trial for metastatic colon cancer. Clearly there is a need to increase treatment alternatives. Our recent work on WNT signaling has determined that the Wnt/?-catenin pathway directs cancer metabolism, promoting aerobic glycolysis and angiogenesis through upregulated expression of glycolysis genes including Pyruvate Dehydrogenase Kinase 1 (PDK1) and SLC16A1/MCT-1. We also discovered that Wnt signaling activity and glycolysis are heterogeneous in xenografts and patient tumors. Tumor heterogeneity is a recognized hallmark of cancer, and it is thought to derive from intracellular reprogramming of metabolic and signaling networks, a flexibility that helps cancer cells adapt to fluctuating nutrient availability and a stressful environment. The Wnt and glycolytic heterogeneity we observe in xenografted colon tumors presents as an array of cell clusters (5-7 cells, ~30m center-to-center) at the proliferating, non-necrotic periphery of tumors. We also observe tumor heterogeneity on a cell-by-cell basis using single cell sequencing analysis. The emergence of a regular array of cell clusters in xenografted tumors suggests that the heterogeneity is patterned and regulated. We therefore developed a mathematical model that recreates this pattern at the leading edge of tumors and Wnt signaling lies at the core of this model. We have tested several model predictions about metabolic patterning and gene expression and the results support the validity of the model. However, an important prediction has not been tested - the one most relevant to developing new ideas for cancer therapy. In silico modeling of tumor growth suggests that a combination of anti-Wnt and anti-PDK (glycolysis) drugs should act synergistically to repress tumor growth. Therefore, the project proposed here is focused on testing this prediction. If this prediction is correct, it would point to a new Achilles Heel for targeting in tumors. In Aim I we will test whether anti- Wnt/anti-PDK combination therapy will synergistically suppress colon cancer xenograft growth using drugs that are currently in clinical trials. In Aim II we will assess how tumor and mouse stroma respond to the single and combination therapy regimen using single cell RNA sequencing (scRNAseq), bulk RNAseq and immunohistochemistry. This short-term, highly feasible project is a necessary step towards a larger scope study focused on cancer metabolism with human PDX and primary patient tissue.